Application of PAD4 inhibitor to preparation of medicine for preventing and treating peripheral neuropathy caused by chemotherapy medicine

Abstract

The invention provides application of a PAD4 inhibitor to preparation of a medicine for preventing and treating peripheral nerve injury caused by chemotherapy medicines. Experiments find that the PAD4 inhibitor has a protective effect on peripheral nerve cell toxicity caused by chemotherapeutic drugs, and particularly has a protective effect on neurotoxicity of ND7 / 23 cells induced by oxaliplatin; on an animal model, the PAD4 inhibitor has a protective effect on oxaliplatin-induced mouse peripheral neurotoxicity. The PAD4 inhibitor can be used as an adjuvant of a chemotherapeutic drug for cancer treatment, and has an effect of relieving neurotoxicity caused by chemotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to use of a PAD4 inhibitor in preparing a drug for preventing and treating peripheral neuropathy induced by chemotherapeutic drugs. Background Art

[0002] CIPN (chemotherapy-induced peripheral neuropathy, CIPN) is a common adverse reaction caused by motor, sensory and autonomic nerve damage caused by anti-tumor drugs, among which sensory dysfunction is the most prominent. Typical symptoms include symmetrical numbness of the limbs, loss of proprioception, obvious pins and needles, hyperalgesia or ectopic pain in the hands and feet. In 2014, the American Society of Clinical Oncology (ASCO) specifically released guidelines for the prevention and treatment of chemotherapy-induced peripheral neuropathy CIPN, pointing out that "CIPN is a very important adverse reaction event that needs our attention, but there is no effective treatment for CIPN today." CIPN-related anti-tumor drugs include platinum drugs (such as carboplatin, cisplatin and oxaliplatin), taxane drugs (such as paclitaxel and docetaxel), eboxilone drugs (such as isabendone), vincristine alkaloids (such as vincristine and vinblastine), bortezomib and thalidomide. CIPN limits the improvement of chemotherapy drug dosage and efficacy to a certain extent, and is the main dose-limiting toxicity of chemotherapy drugs.

[0003] In recent years, how to prevent and alleviate chemotherapy-induced neurotoxicity has become a consensus problem in the field of cancer drug treatment. Platinum drugs cause CIPN mainly by damaging the dorsal ganglion, causing neuronal apoptosis and thus causing neurotoxicity, and its mechanism is mainly related to oxidative stress. Based on the pathogenesis of CIPN, a large number of compounds have been developed to prevent or treat CIPN by blocking ion channels, targeting inflammatory cytokines and anti-oxidative stress. Glutathione and mangafodipir are promising for the prevention of CIPN, and duloxetine is expected to be effective for CIPN intervention, but the mechanism of these drugs in preventing and intervening in CIPN has not yet been elucidated; while drugs such as calcium magnesium injection, venlafaxine, minocycline hydrochloride and gabapentin have limited efficacy in preventing and alleviating CIPN; erythropoietin, menthol and aminofastin are accompanied by a variety of adverse reactions during use.

[0004] PAD4 is a member of the peptidyl arginine deiminase (PAD) family of enzymes that catalyze the citrullination of arginine to citrulline within a peptide sequence. PAD4 is responsible for the deimination or citrullination of a variety of proteins in vitro and in vivo, with different functional responses as a result in various diseases.

[0005] PAD4 inhibitors can also be used to reduce pathological neutrophil activity in a variety of diseases. Studies have shown that the process of NETs (neutrophil extracellular traps, a kind of innate defense mechanism, neutrophils can fix and kill pathogens by it) formation is related to tissue protein citrullination and is lacking in PAD4 knockout mice. Therefore, PAD4 inhibitors may be suitable for diseases in which NET formation in tissues contributes to local damage and disease pathology. Such diseases include but are not limited to small vessel inflammation, systemic lupus erythematosus, ulcerative colitis, cystic fibrosis, asthma, deep vein thrombosis, periodontitis, sepsis, appendicitis, type 2 diabetes and stroke. In addition, evidence shows that NET may contribute to the pathology in diseases such as cutaneous lupus erythematosus and psoriasis that affect the skin, so when applied by systemic or skin routes, PAD4 inhibitors may show the benefits of antagonizing NET skin diseases. PAD4 inhibitors may affect other functions in neutrophils and have a wider applicability to neutrophil diseases.

[0006] Our results suggest that the release of granule proteins and chromatin by neutrophils in the peripheral nervous system after chemotherapy can trigger NETs. Therefore, PAD4 inhibitors may be suitable for diseases where NET formation in peripheral nerve tissue promotes local injury and disease pathology, and are potential therapeutic drugs for CIPN. Summary of the invention

[0007] The purpose of the present invention is to provide a drug for preparing a drug for preventing and treating peripheral nerve damage caused by chemotherapy drugs, so as to improve the peripheral nerve cell toxicity caused by chemotherapy drugs.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention discloses the use of a PAD4 inhibitor for preparing a drug for preventing and treating peripheral nerve damage induced by chemotherapeutic drugs.

[0010] Preferably, the chemotherapeutic drug is a cancer chemotherapeutic drug.

[0011] More preferably, the cancer chemotherapy drug is selected from one or more of platinum chemotherapy drugs, taxane chemotherapy drugs, vincristine chemotherapy drugs, proteasome inhibitors and their derivatives.

[0012] In some embodiments, the platinum chemotherapy drug is one or more of cisplatin, carboplatin, cyclothiaplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0013] In other embodiments, the taxane chemotherapy drug is paclitaxel.

[0014] In other embodiments, the vincristine chemotherapy drug includes one or more of vinblastine and vincristine.

[0015] In other embodiments, the proteasome inhibitor chemotherapy drug is bortezomib.

[0016] In the above scheme, the symptoms of peripheral nerve injury include one or more of the following: numbness and pain in the skin of the extremities, glove-like or stocking-like sensation, cold skin, paresthesia, a sense of ants, weakness in holding objects, a feeling of walking on cotton, burning pain, shooting pain, and knife-like pain.

[0017] In a second aspect, the present invention provides a medicament for preventing and treating peripheral nerve damage induced by chemotherapeutic drugs, the medicament comprising a PAD4 inhibitor and a pharmaceutically acceptable carrier.

[0018] In a third aspect, the present invention further provides a method for preparing a drug for preventing and treating peripheral nerve damage induced by chemotherapeutic drugs, the method comprising mixing a PAD4 inhibitor and a pharmaceutically acceptable carrier in proportion.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides the use of PAD4 inhibitors in the preparation of drugs for preventing and treating peripheral nerve damage caused by chemotherapy drugs, and further experiments have found that PAD4 inhibitors interfere with the formation of NETs in the peripheral nervous system induced by oxaliplatin; in animal models, PAD4 inhibitors have a protective effect on oxaliplatin-induced peripheral neurotoxicity in mice. The drug PAD4 inhibitor of the present invention can be used as an adjuvant of chemotherapy drugs for cancer treatment, and has a mitigating effect on neurotoxicity caused by chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a chemical structure diagram of a PAD4 inhibitor;

[0021] Figure 2 The effect of PAD4 inhibitor on the total content of neutrophil enzyme MPO induced by oxaliplatin, paclitaxel and bortezomib;

[0022] Figure 3 The effect of PAD4 inhibitor on the body weight of OIPN model mice;

[0023] Figure 4 The effect of PAD4 inhibitor on mechanical pain threshold of OIPN mice;

[0024] Figure 5 The effect of PAD4 inhibitor on cold pain scores in OIPN mice;

[0025] Figure 6This is the effect of PAD4 inhibitor on the mechanical pain threshold of PIPN and BIPN mice.

[0026] Note: Data are expressed as mean ± standard deviation (n = 3). Figure 1-4 One-way ANOVA was performed, followed by Tukey's post hoc comparison. *P<0.05, **P<0.01 compared with the blank group; #P<0.05, ##P<0.01 compared with the model group. DETAILED DESCRIPTION

[0027] The inventors have found that PAD4 inhibitors have the effect of preventing the activation of NETs in the peripheral nervous system caused by chemotherapy drugs. Experiments have found that PAD4 inhibitors interfere with the formation of NETs in the peripheral nervous system caused by chemotherapy drugs, especially the formation of NETs in the peripheral nervous system induced by platinum chemotherapy drugs (such as oxaliplatin) has a more clear interference effect; in animal models, PAD4 inhibitors have a protective effect on peripheral neurotoxicity of mice induced by platinum chemotherapy drugs. Therefore, the present invention provides a pharmaceutical composition comprising a PAD4 inhibitor, and the PAD4 inhibitor in the drug of the present invention can be used as an adjuvant for chemotherapy drugs for cancer treatment, and has a mitigating effect on neurotoxicity caused by chemotherapy.

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicating the orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0030] In this specification, the reference to "an implementation method" or "an implementation method" means that specific features, structures and attributes are incorporated into the implementation method. Among them, these characteristics and structures are included in at least one implementation method. Therefore, the expression "in an implementation method" or "in an implementation method" does not necessarily mean that it is equivalent to the same implementation method. In addition, in one or more implementation methods, specific characteristics, structures and attributes can be combined and used in any manner as required. It should be pointed out that the singular forms "one", "a kind" and "the one" used in this specification and the attached claims actually include the plural meaning, unless the specific content expressly indicates otherwise. Finally, the word "or" usually has its literal meaning, that is, "and / or", unless the context has other special provisions.

[0031] As used herein, the term "peripheral nerves" generally refers to those nerves that branch out from the spinal cord and extend to various parts of the body. Nerve repair is more common in the peripheral nervous system than in the central nervous system.

[0032] As used herein, chemotherapy-induced peripheral nerve damage (CIPN) is a type of nerve damage caused by chemotherapy drugs, usually manifested as paresthesia, pain, numbness, tingling and other symptoms. Chemotherapy drugs affect the normal function of nerve cells, resulting in impaired nerve conduction, which in turn causes peripheral nerve damage. Common chemotherapy drugs include taxanes (such as paclitaxel), platinum drugs (such as oxaliplatin), enzyme inhibitors (such as vincristine), etc. Peripheral nerve damage may affect the patient's quality of life, so the occurrence and development of CIPN needs to be closely monitored and managed during chemotherapy.

[0033] The terms "therapeutic", "treatment" or "relief", "relief" in the present invention not only refer to medical methods used to treat diseases (such as slowing down or curing symptoms), stop (or reduce) the progression of diagnosed conditions, and prevent peripheral nerve damage, but also cover measures to prevent the slow regeneration process of peripheral nerves. Therefore, the patients treated include people with peripheral nerve damage symptoms; and those who are susceptible to peripheral nerve damage. The ideal medical effect may include but is not limited to symptom relief, reduced degree of nerve tissue damage, maintaining a stable (i.e., not deteriorating) state of nerve damage, slowing down or preventing the development of damage, recovery of nerve tissue (whether partial or complete) or even regeneration. The people treated include people who have suffered from peripheral nerve damage or suspected of having such a disease, as well as people who may be susceptible to such damage. These people may need to prevent this from happening for various reasons, or avoid greater damage. In addition, the population that needs special treatment also includes animals whose nerve regeneration activity ability degenerates with age.

[0034] In the present invention, the term "therapeutically effective amount" refers to the amount of small organic molecules or other drugs that are effective for "treating" a disease or condition in a subject or mammal. In the case of peripheral nerve injury or trauma (e.g., sciatic nerve trauma), a therapeutically effective amount of a drug can promote axon regeneration of sensory neurons, epidermal innervation, and functional recovery of neurons after nerve injury in subjects with nerve regeneration decline, by increasing, for example, the proliferation, differentiation, migration and / or survival of neural stem cells / precursor cells; reducing, delaying or stopping the reduction of nerve cells; inhibiting, for example, suppressing, delaying, preventing, stopping or reversing the reduction of nerve cells; increasing the number, density and / or concentration of nerve cells; changes in the morphology or function of nerve cells; or changes in the interaction between nerve cells; alleviating one or more symptoms associated with nerve injury to a certain extent, such as pain; improving the quality of life; or a combination of these effects.

[0035] The treatment subjects of the present invention include individuals, animals and mammals. These animals include but are not limited to humans, household pets, livestock, animals in wild animals, competitive animals and pets, such as dogs, cats, hamsters, rabbits, rats, mice, horses, cattle, cows and bears.

[0036] The administration of the compounds of the present invention can be oral, but, as an alternative to oral drugs, in certain situations, other routes of administration can be applied to the technology and application scenarios described herein, such as intravenous administration. There are many routes of intravenous administration, such as subcutaneous, intravenous, muscle, joint, synovial membrane, sternum, sheath, liver, lesions, and intracranial sites. For example, the compound can also be administered by intravenous, intramuscular, sheath or subcutaneous injection. In some applications, in order to achieve the purpose of oral drugs, the dosage of the compound can be reduced to a level suitable for intravenous administration, and then achieved by injection.

[0037] According to the usage and functions described herein, there are other suitable scenarios where the compound can be applied for administration, such as: sublingual, inside the cheek (such as applying a film or other mixture that can be dissolved in the mouth under the tongue or inside the cheek), eyes (such as as eye drops), ears (such as through ear drops), oral inhalation (such as by blowing or atomization), skin or local area (such as by applying emulsion or lotion) or directly applied to the skin (such as through a patch). In addition to oral administration, it can also be delivered through other intestinal administration methods such as vaginal and rectal administration, such as through ointments, suppositories or enemas.

[0038] In practical applications, the operations and purposes involved in this study are usually achieved by adding effective compounds (such as oral administration) to achieve the goal of alleviating symptoms. The specific effective dose range used is 0.05 mg / kg to 20 mg / kg, including 0.05 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.05 mg / kg to 5 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.05 mg / kg to 2 mg / kg, 0.1 mg / kg to 2 mg / kg, and any dose range with any interval set at 0.05 mg / kg and 20 mg / kg or other intermediate values ​​as endpoints. In some cases, this strategy may require the use of a compound with a dose range of 0.1-1 mg / kg, or a compound with a dose range of 0.2-0.5 mg / kg. Likewise, the methods or uses may involve administering a dose of the compound in some cases in the range of 0.05-20 mg / kg, or in some cases in the range of 1-10 mg / kg.

[0039] In some embodiments, the methods and uses described herein, such as methods or uses for treating pain in a subject (e.g., a human) in need thereof, are achieved by administering (e.g., orally) a therapeutically effective amount of a compound per day, such as 5 to 1000 mg of a compound per day, such as 5 to 500 mg or 5 to 250 mg of a compound per day. For example, the method or use may include administering (e.g., orally) about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135mg, about 140mg, about 145mg, about 150mg, about 155mg, about 160mg, about 165mg, about 170mg, about 175mg, about 180mg, about 185mg, about 190mg, about 195mg, about 200mg, about 205mg, about 210mg, about 215mg, about 220mg, about 225mg, about 230mg, about 235mg, about 240mg, about 245mg, about 250mg, about 255mg, about 260mg, about 265mg, about 270mg, about 275mg, about 280mg, about 285mg, about 290mg, about 295mg, about 300mg, about 305mg, about 310mg, about 315mg, about 320mg, about 325mg, about 330mg, about 335mg, about 340, about 345mg, about 350mg, about 355mg, about 360mg, about 365mg, about 370mg, about 375mg, about 380mg, about 385mg, about 390mg, about 395mg, about 400mg, About 405mg, about 410mg, about 415mg, about 420mg, about 425mg, about 430mg, about 435mg, about 440mg, about 445mg, about 450mg, about 455mg, about 460mg, about 465mg, about 470mg, about 475mg, about 480mg, about 485mg, about 490mg, about 495mg, about 500mg or about 1000mg of a compound, or daily administration (e.g., oral) by using two of the above quantities as any number range created by endpoints. In some aspects, the method or uses include oral administration of 10 to 200mg of a compound to a subject (e.g., a person) every day, for example, 10, 15, 20, 25, 30, 35 or 40mg to 75, 100, 125, 150, 175 or 200mg of a compound per day, including 20 to 150mg per day.In some aspects, the subject (e.g., human) is orally administered with a compound comprising 50, 75, 100 or 125 mg per day, for example, 100 mg per day. In some cases, the daily dose of the compound may need to be achieved by multiple doses (e.g., oral), such as 5 times 20 mg, 4 times 25 mg, 3 times 33.3 mg or 2 times 50 mg, etc., each time. For example, a daily dose of 100 mg can be allocated to different dosing regimens such as 5 times 20 mg, 4 times 25 mg, 3 times 33.3 mg or 2 times 50 mg per day. In some methods, the daily dose of the compound can also be regarded as a single dose for use (e.g., oral). For example, the daily dose of a compound can be allocated to take 5 mg, 10 mg, 15 mg, 20 mg, 25 mg or 30 mg alone, or a dose in 5 mg, 10 mg, 15 mg, 20 mg, 25 mg or 30 mg, such as taking 5 mg once a day or taking 10 mg once a day, etc. Also, any dose of the compound discussed in the previous section may be distributed in a single dosage unit or multiple dosage unit dosage form, such as two, three or four unit dosage forms.

[0040] In some embodiments, the methods and uses described herein for treatment by administering (e.g., orally) a compound include administering the compound at the same time as, before, or after chemotherapy, for example, 0.5h, 1h, 1.5h, 2h before chemotherapy, or 0.5h, 1h, 1.5h, 2h after chemotherapy; it can also be administered 1 day, 2 days, 3 days before chemotherapy, 1 day, 2 days, 3 days after chemotherapy, etc.

[0041] The drug complex of the present invention covers various forms (not limited): injection, tablets, capsules, gas release agents, patches, films, pills, external coatings, controlled release or slow release or nanoparticles. This drug complex contains a primary compound or a pharmacologically acceptable salt thereof that is safe and pharmacologically acceptable within its effective dose range, and a pharmacologically acceptable carrier. The so-called "effective dose" means that the content of the drug can significantly improve the symptoms without causing serious side effects. Generally speaking, the drug complex contains 1-2000 mg of the primary compound / dose, and even better, it contains 10-1000 mg of the primary compound / dose. In the best case, the so-called "one unit" is a capsule or tablet. "Acceptable carrier" refers to one or more compatible solid or liquid filling materials suitable for the human body, which must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the various components in the drug complex can be mixed with the primary compound and between them, and will not significantly reduce the efficacy of the compound. For example, some raw materials of acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween 80) and colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0042] There are many types of solid preparations for oral administration, such as capsules, tablets, pills, powders and granules. The active ingredients in these preparations are mixed with some common inactive auxiliary agents, such as sodium citrate or dicalcium phosphate, or with other specific ingredients: (i) fillers or thickeners, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (ii) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (iii) humectants, such as glycerol; (iv) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (v) sustained release agents, such as paraffin; (vi) absorption promoters, such as quaternary ammonium compounds; (vii) wetting agents, such as cetyl alcohol and glyceryl monostearate; (viii) adsorbents, such as kaolin; (ix) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. Buffers may also be added to the three preparations of capsules, tablets and pills. Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be obtained by coating and outer shelling methods, such as enteric coatings and other common materials. These preparations may also contain opaque fillers, and in the preparations, the active compound or its release mode can be released in a specific part of the digestive tract. Buffers may also be added to capsules, tablets and pills. Oral liquid preparations include emulsions, solutions, suspensions, syrups or tinctures accepted in the medical field. In addition, liquid preparations may also contain inactive diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition, in addition to these inactive diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices. The suspension may also contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances. In the composition for parenteral injection, a physiologically acceptable sterilized water or anhydrous solution, dispersion, suspension or emulsion and a sterile powder for re-dissolving into a sterile injectable solution or dispersion may be contained. Suitable as water, ethanol, polyols and their appropriate mixtures. In the pharmaceutical formulation for local use, ointments, powders, patches, sprays and inhalants are included. Under sterile conditions, the active ingredient is mixed with a physiologically acceptable carrier, any preservative, buffer, or, if necessary, a propellant. This treatment method can be used independently or in combination with other treatment methods or drugs.

[0043] Example 1: Chemical structure diagram of PAD4 inhibitors.

[0044] Example 2: Effect of PAD4 inhibitor on the total content of neutrophil enzyme MPO induced by oxaliplatin, paclitaxel and bortezomib.

[0045] 1. Experimental materials:

[0046] Experimental drugs: Oxaliplatin (CAS No.: 61825-94-3) and PAD4 inhibitor GSK484 (CAS No.: HY-100514) were purchased from MCE. Paclitaxel (CAS No.: HWG53914) was purchased from Beijing Huawei Ruike Chemical Co., Ltd., and bortezomib (CAS No.: 179324-69-7) was purchased from Taoshu Biological. Oxaliplatin was prepared with 5% glucose solution, and PAD4 inhibitor GSK484 was prepared with pure water.

[0047] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0048] 2. Experimental methods:

[0049] (1) The experimental animals were randomly divided into a blank group, a model group, and a PAD4 inhibitor GSK484 group.

[0050] Blank group: intraperitoneal injection of solvent;

[0051] OIPN model group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, and the drug was administered for 5 days and stopped for 5 days, for a total of two cycles of 20 days; pure water was gavage at a dose of 4 ml / kg, and the drug was administered every day.

[0052] PIPN model group: intraperitoneal injection of paclitaxel solution 10 mg / kg (once on the 1st, 3rd, 5th and 7th day, 4 injections in total for 4 days). Oral administration of pure water at a dose of 4 ml / kg was continued every day.

[0053] BIPN model group: Bortezomib 1 mg / kg was injected intraperitoneally (once on the 1st, 3rd, 5th and 7th day, 4 times in total for 4 days). Pure water was administered intragastrically at a dose of 4 ml / kg for consecutive days.

[0054] PAD4 inhibitor GSK484 group: 4 mg / kg of PAD4 inhibitor GSK484 solution was intraperitoneally injected 2 hours in advance, and then 3 mg / kg of oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg. PAD4 inhibitors need to be administered every day, and oxaliplatin is administered for 5 days and stopped for 5 days, for a total of two cycles of 20 days.

[0055] (2) The first day of drug treatment was designated as Day 1. On Day 21, mice were dissected and DRGs were obtained. The DRG homogenate was placed in 50 mM potassium dihydrogen phosphate buffer, centrifuged, and suspended in 0.5% hexadecyltrimethylammonium bromide potassium dihydrogen phosphate buffer. The suspension was subjected to three freeze-thaw cycles and ultrasonic treatment for 30 s in liquid nitrogen. After centrifugation, 40 mL of the supernatant was incubated with 100 μL of tetramethylbenzidine solution, and the reaction was stopped with 100 μL of 2N HCl. The absorbance was measured at 450 nm using a full-wavelength multifunctional microplate reader (model: Varioskan flash, purchased from Thermo Scientific, USA).

[0056] 3. Experimental results:

[0057] The results are as follows Figure 2 As shown in the figure, it can be seen that the total content of neutrophil enzyme MPO was significantly increased in the oxaliplatin, paclitaxel, and bortezomib groups. The total content of neutrophil enzyme MPO decreased when oxaliplatin, paclitaxel, bortezomib and 50nM PAD4 inhibitor GSK484 were co-treated, indicating that the PAD4 inhibitor GSK484 inhibited the increase in the total content of neutrophil enzyme MPO caused by oxaliplatin, paclitaxel, and bortezomib. One-way ANOVA was used, followed by Tukey's post hoc comparison. Compared with the blank group, *P<0.05, **P<0.01; compared with the oxaliplatin group, #P<0.05, ##P<0.01.

[0058] Example 3: PAD4 inhibitors have a protective effect on oxaliplatin-induced peripheral neuropathy in mice.

[0059] 1. Experimental materials:

[0060] Experimental drugs: Oxaliplatin (CAS No.: 61825-94-3) and PAD4 inhibitor GSK484 (CAS No.: HY-100514) were purchased from MCE. Oxaliplatin was prepared with 5% glucose solution, and PAD4 inhibitor was prepared with pure water.

[0061] Instruments: Electronic von Frey analgesia meter, Plantar Test (Hargreaves method) Glass stands, and Hot Cold Plate Analgesia Meter for Mice and Rats were all purchased from IITC (IITC Inc. Life Science, Woodland Hills, CA, USA).

[0062] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0063] 2. Experimental methods:

[0064] (1) The experimental animals were randomly divided into three groups: blank group, model group and PAD4 inhibitor GSK484 group.

[0065] Blank group: intraperitoneal injection of solvent;

[0066] Model group: 3 mg / kg oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg, 5 days of administration and 5 days of rest, for a total of two cycles of 20 days; pure water was gavage at a dose of 4 ml / kg, and the drug was administered every day.

[0067] PAD4 inhibitor GSK484 group: 4 mg / kg of PAD4 inhibitor GSK484 solution was intraperitoneally injected 2 hours in advance, and then 3 mg / kg of oxaliplatin 5% glucose solution was intraperitoneally injected at a dose of 5 ml / kg. PAD4 inhibitors need to be administered every day, and oxaliplatin is administered for 5 days and stopped for 5 days, for a total of two cycles of 20 days.

[0068] (2) The first day of drug treatment was designated as Day 1, and the weight and behavioral tests (mechanical pain test and cold pain test) of mice were performed on Days 0, 6, 11, 16, and 21.

[0069] (A) Body weight: The body weight of mice was measured at the same time point every day.

[0070] (B) Experimental measurement of mechanical pain sensation in mice: Referring to the method of Meng Zhao et al. (KIMeng Zhao, Saki Nakamura, Takayu K1 nakagawa, Shuji Kaneko, Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice, Molecular Pain. 8 (2012) 55), the mechanical withdrawal threshold (von Frey test) was used to evaluate the mechanical allodynia of mice. The mice were placed in a (20*17*13cm) plexiglass box, and the bottom contact surface of the mouse toes was a metal mesh. After the mice adapted to the measurement box environment for 15 minutes and were at rest, the soles of the feet of the mice were vertically stimulated with von Frey fiber filaments. After the mice showed a withdrawal reaction, the electronic display stress value was recorded. Each mouse was measured 10 times.

[0071] (C) Mouse cold pain test: Before the measurement, the mice were placed in the measurement environment to adapt for 30 minutes. During the test, the mice were placed on a cold plate at 5°C and covered with a transparent plexiglass box. The measurement time was 60 seconds. During the measurement time, the escape behavior of the mice was recorded and scored: 0 points = no reaction; 1 point = mild cold escape reaction, such as lifting the hind legs or walking backwards; 2 points = strong cold escape reaction, such as jumping up, etc. The total score within the measurement time of 60 seconds was recorded. Each mouse was measured 3 times and the average was taken.

[0072] 3. Experimental results:

[0073] The results are as follows Figure 3 , Figure 4 , Figure 5 As shown, according to the diagram:

[0074] (A) Effects on the body weight of mice: Figure 3 As shown in the figure, the weight of mice in the blank group increased over time. The mice in the model group showed a significant weight loss due to gastrointestinal adverse reactions caused by oxaliplatin stimulation. During the withdrawal of oxaliplatin, the weight of mice showed a slow increase. The weight of mice in the PAD4 inhibitor GSK484 group also decreased significantly during the administration of oxaliplatin, and the weight of mice increased after oxaliplatin withdrawal. There was no significant change in the weight of mice in the PAD4 inhibitor GSK484 group compared with the model group.

[0075] (B) Effects on mechanical pain perception in mice: Figure 4As shown in the figure, during the measurement period, the mechanical paw withdrawal threshold of the blank group mice tended to be stable. The mechanical paw withdrawal threshold of the model group mice decreased significantly compared with the control group; compared with the model group, the mechanical pain threshold of the PAD4 inhibitor GSK484 group mice was significantly increased. This indicates that the PAD4 inhibitor GSK484 can reduce the mechanical pain sensitivity of mice caused by oxaliplatin.

[0076] (C) Effects on cold pain perception in mice: Before the experiment, mice in each group were insensitive to cold pain perception. Figure 5 As shown in the figure, after 11 days of the experiment, the cold pain scores of mice in the model group increased significantly compared with those in the blank group; when measured on the 16th and 21st days, the mice in the model group showed obvious cold pain sensitivity compared with the mice in the blank group. When measured from the 11th day to the 21st day, the cold pain scores of mice in the PAD4 inhibitor GSK484 group were lower than those in the model group, and there was a significant difference. This shows that the PAD4 inhibitor GSK484 can reduce the cold pain sensitivity of mice caused by oxaliplatin.

[0077] In summary, in the peripheral nerve injury model caused by oxaliplatin, mice were sensitive to mechanical pain and cold pain, while pre-administration of the PAD4 inhibitor GSK484 reduced the sensitivity of mechanical pain and cold pain in mice. The PAD4 inhibitor GSK484 had a protective effect on peripheral nerve injury caused by oxaliplatin in mice. Two-way ANOVA was used, followed by Tukey's post hoc comparison. The model group was compared with the blank group *P<0.05, **P<0.01; the PAD4 inhibitor GSK484 group was compared with the model group, #P<0.05, ##P<0.01.

[0078] Example 4: PAD4 inhibitor GSK484 has a protective effect on paclitaxel- and bortezomib-induced peripheral neuropathy in mice.

[0079] 1. Experimental materials:

[0080] Experimental drugs: Paclitaxel (CAS No.: HWG53914) was purchased from Beijing Huawei Ruike Chemical Co., Ltd., bortezomib (CAS No.: 179324-69-7) was purchased from Taoshu Biological, and PAD4 inhibitor GSK484 (CAS No.: HY-100514) was purchased from MCE. Paclitaxel was prepared with polyoxyethylene castor oil and anhydrous ethanol, bortezomib was prepared with physiological saline, and PAD4 inhibitor GSK484 was prepared with pure water.

[0081] Instruments: Electronic von Frey analgesia instrument, Plantar Test (Hargreaves method) and Glass stands were purchased from IITC (IITC Inc. Life Science, Woodland Hills, CA, USA).

[0082] Experimental animals: Male C57BL / 6 mice, weighing 20-24g, clean grade, purchased from Shanghai Slake Experimental Animal Center, and kept in the SPF-grade experimental animal room of Jiangsu Institute of Traditional Chinese Medicine. The experimental temperature was controlled at 25±1℃, and free access to drinking water and animal feed was provided. The experiment began after one week of feeding.

[0083] 2. Experimental methods:

[0084] (1) The experimental animals were randomly divided into the following three groups: blank group, PIPN model group and PAD4 inhibitor-PIPN group; blank group, BIPN model group and PAD4 inhibitor-BIPN group.

[0085] Normal group: intraperitoneal injection of solvent;

[0086] PIPN model group: intraperitoneal injection of paclitaxel solution 10 mg / kg (once on the 1st, 3rd, 5th and 7th day, 4 injections in total for 4 days). Oral administration of pure water at a dose of 4 ml / kg was continued every day.

[0087] PAD4 inhibitor-PIPN group: 4 mg / kg of PAD4 inhibitor GSK484 solution was intraperitoneally injected 2 hours in advance, and then 8 mg / kg of paclitaxel solution was intraperitoneally injected. PAD4 inhibitors need to be administered every day, and paclitaxel is injected once on the 1st, 4th, 8th, and 11th day, for a total of 4 injections over 4 days.

[0088] BIPN model group: Bortezomib 1 mg / kg was injected intraperitoneally (once on the 1st, 3rd, 5th and 7th day, 4 times in total for 4 days). Pure water was administered intragastrically at a dose of 4 ml / kg for consecutive days.

[0089] PAD4 inhibitor-BIPN group: 4 mg / kg of PAD4 inhibitor GSK484 solution was intraperitoneally injected 2 hours in advance, and then 1 mg / kg of bortezomib solution was intraperitoneally injected. PAD4 inhibitors need to be administered every day, and bortezomib is injected once on the 1st, 3rd, 5th, and 7th day, for a total of 4 injections over 4 days.

[0090] (2) The first day of drug treatment was designated as Day 1, and mechanical pain tests were performed on mice on Days 0 and 8, respectively.

[0091] (A) Experimental determination of mechanical pain sensation in mice: Referring to the method of Meng Zhao et al. (KIMeng Zhao, Saki Nakamura, Takayu K1 nakagawa, Shuji Kaneko, Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice, Molecular Pain. 8 (2012) 55), the mechanical withdrawal threshold (von Frey test) was used to evaluate the mechanical allodynia of mice. The mice were placed in a (20*17*13cm) plexiglass box, and the bottom contact surface of the mouse toes was a metal mesh. After the mice adapted to the measurement box environment for 15 minutes and were at rest, the soles of the mice's feet were vertically stimulated with von Frey fiber filaments. After the mice showed a withdrawal reaction, the electronic display stress value was recorded. Each mouse was measured 10 times.

[0092] 3. Experimental results:

[0093] The results are as follows Figure 6 As shown, according to the diagram:

[0094] (A) Effects on mechanical pain perception in mice: Figure 6 As shown in the figure, during the measurement period, the mechanical paw withdrawal threshold of the blank group mice tended to be stable. The mechanical paw withdrawal threshold of the PIPN and BIPN model groups mice decreased significantly compared with the control group; compared with the PIPN and BIPN model groups, the mechanical pain threshold of the PAD4 inhibitor GSK484 group mice increased significantly. This indicates that the PAD4 inhibitor GSK484 can reduce the mechanical pain sensitivity of mice caused by oxaliplatin.

[0095] In summary, in the peripheral nerve injury model caused by paclitaxel and bortezomib, mice were sensitive to mechanical pain, and pre-administration of the PAD4 inhibitor GSK484 reduced the mechanical pain sensitivity of mice. The PAD4 inhibitor had a protective effect on the peripheral nerve injury of mice caused by paclitaxel and bortezomib. Two-way ANOVA was used, followed by Tukey's post hoc comparison. The model group was compared with the blank group *P<0.05, **P<0.01; the PAD4 inhibitor GSK484 group was compared with the PIPN and BIPN model groups, #P<0.05, ##P<0.01.

[0096] It should be understood that although the embodiments of the present invention use the PAD4 inhibitor GSK484 as an example to illustrate the use of PAD4 inhibitors for preventing and treating peripheral nerve damage caused by chemotherapy drugs, it should be understood that the above embodiments are for illustrative purposes only. After the technical personnel in the industry read the documents of the present invention and understand the principles of the present invention, they can easily understand that other PAD4 inhibitors can also be used for the above purposes, such as other PAD4 inhibitors such as GSK199, PAD4 inhibitors JBI-589, 9323, etc., and therefore other PAD4 inhibitors should also be included in the scope of protection of the present invention.

[0097] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0098] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Use of PAD4 inhibitors for preparing drugs for preventing and treating peripheral nerve damage caused by chemotherapy drugs.

2. The use according to claim 1, characterized in that The chemotherapy drug is a cancer chemotherapy drug.

3. The use according to claim 2, characterized in that The cancer chemotherapy drug is selected from one or more of platinum chemotherapy drugs, taxane chemotherapy drugs, vincristine chemotherapy drugs, proteasome inhibitor chemotherapy drugs bortezomib and their derivatives.

4. The use according to claim 3, characterized in that The platinum chemotherapy drug is one or more of cisplatin, carboplatin, cyclothioplatin, nedaplatin, oxaliplatin, and lobaplatin.

5. The use according to claim 3, characterized in that The taxane chemotherapy drug is one or more of paclitaxel and docetaxel.

6. The use according to claim 3, characterized in that The vincristine chemotherapy drugs include one or more of vinblastine and vincristine.

7. The use according to claim 1, characterized in that The symptoms of peripheral nerve injury include one or more of the following: numbness and pain in the skin of the extremities, a glove or stocking-like sensation, cold skin, paresthesia, a sense of ants, weakness in holding objects, a feeling of walking on cotton, burning pain, shooting pain, and knife-like pain.

8. A drug for preventing and treating peripheral nerve damage caused by chemotherapy drugs, characterized in that: The medicament comprises a PAD4 inhibitor and a pharmaceutically acceptable carrier.

9. A method for preparing a drug for preventing and treating peripheral nerve damage caused by chemotherapeutic drugs, characterized in that: The method comprises mixing a PAD4 inhibitor and a pharmaceutically acceptable carrier in proportion.

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